The mechanism of heat stress-induced injury in the immature oocytes of zebrafish (Danio rerio).

Immature zebrafish oocytes are highly susceptible to high temperatures, making it difficult to warm cryopreserved oocytes rapidly. In the present study, we aimed to investigate whether thermosensitive channels, lipid mediators, and ferroptosis are involved in heat stress-induced injury in immature zebrafish oocytes. Oocytes were injected with inhibitors of a heat-sensitive channel (TRPV1) and multiple enzymes-cytosolic phospholipase A₂α (cPLA₂α), cyclooxygenases (COXs), arachidonate lipoxygenase 5 (ALOX5), and lysophosphatidylcholine acyltransferase 2 (LPCAT2). In addition, a ferroptosis-specific inhibitor was administered. The oocytes were then warmed at 45°C for 15 min, incubated at 25°C for 2 h, and then stained with propidium iodide. When the control oocytes were warmed at 45°C for 15 min, their survival was low (1%-8%). However, the survival of oocytes injected with the TRPV1-specific inhibitor markedly improved (40%), suggesting that TRPV1 activation triggers heat stress injury in oocytes. When a cPLA₂α-specific inhibitor was injected, survival of oocytes after warming significantly improved (30%), suggesting that lipid mediators or ferroptosis are involved in heat stress-induced injury in oocytes. In contrast, survival either slightly improved or did not improve when oocytes were injected with specific inhibitors of COXs, ALOX5, and LPCAT2 (16%, 8%, and 3%, respectively). Notably, the ferroptosis-specific inhibitor markedly improved oocyte survival (60%). These results may facilitate methodological advancements in fish oocyte cryopreservation. Additionally, they suggest that ferroptosis is involved in heat stress-induced injury in immature zebrafish oocytes, following TRPV1 activation and subsequent cPLA₂α activation.